JP7302865B2 - Method for inducing immature oocytes and method for producing mature oocytes - Google Patents

Description

The present invention relates to a method for deriving immature oocytes and a method for producing mature oocytes.

In mammals, totipotency, defined as the ontogenic potential from a single cell, is a special property of single cells. However, the mechanism that forms such omnipotence has been difficult despite strong social demand in fields such as fertility treatment. The reason for this is that the oogenesis process that proceeds in the fetal ovary cannot be reproduced in vitro.

The inventors have so far developed an in vitro culture system for reconstructing oocytes from mouse pluripotent stem cells. Specifically, mouse ES cells or iPS cells are differentiated into primordial germ cell-like cells (PGCLCs) using a medium containing humoral factors such as BMP4, and the resulting PGCLCs are mixed with ovarian somatic cells and regenerated. Create a construct ovary. Then, the period from PGCLCs to formation of the second meiotic metaphase egg in the reconstructed ovary is divided into three periods, "in vitro differentiation period", "in vitro growth period" and "in vitro maturation period", each In the culture period of , we have established optimal culture conditions for obtaining secondary follicles, germinal vesicle stage eggs, and second meiotic metaphase eggs (see, for example, Non-Patent Document 1, etc.).

Hikabe O et al., “Reconstitution in vitro of the entire cycle of the mouse female germ line.”, Nature, Vol. 539, p299-303, 2016.

However, the method described in Non-Patent Document 1 requires a period of about three to four weeks in order to obtain oocytes of primordial follicles from mouse pluripotent stem cells. In mammals, it is expected that one year or more of culture will be required to produce oocytes from pluripotent stem cells in an in vitro culture system.

The present invention has been made in view of the above circumstances, and is capable of inducing immature oocytes from cells having the ability to differentiate into oocytes such as pluripotent stem cells in a shorter period of culture than in the past. provide a way to Also provided is a method for easily producing mature oocytes from cells having the ability to differentiate into oocytes, such as pluripotent stem cells, in a shorter period of culture than conventionally.

As a result of intensive research to achieve the above object, the inventors have found that a specific gene involved in the formation of oocytes is introduced into pluripotent stem cells and cultured for a short period of time of 5 days or more and 10 days or less. The inventors have found that differentiation can be induced into immature oocytes in , and have completed the present invention.

That is, the present invention includes the following aspects.
The method for inducing immature oocytes according to the first aspect of the present invention comprises four types of genes consisting of FIGLA, NOBOX, LHX8 and TBPL2, or their transcripts or expressed proteins, in pluripotent stem cells and epiblast-like cells. and introducing into at least one cell selected from the group consisting of primordial germ cells.
The method for inducing immature oocytes according to the first aspect may include introducing four types of genes consisting of FIGLA, NOBOX, LHX8 and TBPL2 into the cells.
The method for inducing an immature oocyte according to the first aspect may further include introducing a STAT3 gene, or its transcript or expressed protein into the cell.
The method for inducing immature oocytes according to the first aspect further comprises introducing into the cell one or more genes selected from the group consisting of SOHLH1, SUB1 and DYNLL1, or transcripts or expressed proteins thereof. may contain.
The method for inducing immature oocytes according to the first aspect may further include introducing three types of genes consisting of SOHLH1, SUB1 and DYNLL1 into the cells.
The cells may be pluripotent stem cells.
In the method for inducing immature oocytes according to the first aspect, the expression of the gene is controlled to be induced by the presence of an expression inducer,
Proliferating said cells after said introduction;
After the growth, the method may further include adding the expression-inducing substance to the medium to induce the expression of the gene.

A method for producing a mature oocyte according to the second aspect of the present invention comprises:
introducing four types of genes consisting of FIGLA, NOBOX, LHX8 and TBPL2, or their transcripts or expressed proteins into at least one cell selected from the group consisting of pluripotent stem cells and primordial germ cells;
co-culturing the cells and ovarian somatic cells after introduction;
including.
The method for producing a mature oocyte according to the second aspect may further include introducing a STAT3 gene, or its transcript or expressed protein into the cell.

According to the method for inducing immature oocytes of the above aspect, immature oocytes are induced from cells having the ability to differentiate into oocytes such as pluripotent stem cells in a shorter period of culture than conventionally. be able to. According to the method for producing mature oocytes of the above aspect, a large amount of mature oocytes are produced from cells having the ability to differentiate into oocytes such as pluripotent stem cells in a shorter period of culture than conventionally. be able to.

1 shows bright field images and fluorescent images (magnification: 200 times) of immature oocytes induced from mouse ES cells in Example 1. FIG. 1 is a fluorescence image (magnification: 8 times) of aggregates composed of pluripotent stem cells into which an oocyte-forming gene has been introduced and ovarian somatic cells at each culture day in Example 1. FIG. At the top, oocytes were visualized by the fluorescence of enhanced cyan fluorescent protein (ECFP) expressed under the control of Stella, a germ cell and oocyte marker. At the bottom, oocytes were visualized by fluorescence of the red fluorescent protein mCherry expressed under the expression control of Nucleoplasmin 2 (Npm2), an activated (mature) oocyte marker. FIG. 2 shows bright-field and fluorescent images (magnification: 8×) of immature oocytes induced from mouse iPS cells in Example 2. FIG. FIG. 10 shows the results of identifying important factors in the oocyte formation gene in Example 3. FIG. In the figure on the left, the vertical axis indicates the sample name, and the horizontal axis indicates the type of oocyte formation gene. White columns indicate that the gene has not been introduced. The middle figure shows the number of oocytes formed from each cell line. The figure on the right shows the oocyte area calculated from the fluorescence area of the blue fluorescent protein CFP, which is expressed downstream of the Stella gene in each sample.

<Method for inducing immature oocytes>
In one embodiment, the present invention selects four types of genes consisting of FIGLA, NOBOX, LHX8 and TBPL2, or their transcripts or expressed proteins, from the group consisting of pluripotent stem cells, epiblast-like cells and primordial germ cells. A method for inducing immature oocytes, comprising introducing into at least one type of cell (hereinafter these cells may be collectively referred to as "cells capable of differentiating into oocytes") .

According to the conventional method, in the case of mice, it takes about a little less than one month to induce differentiation from pluripotent stem cells to immature oocytes in vitro. It took about 11 days to induce differentiation to immature oocytes. On the other hand, in the method for inducing immature oocytes of the present embodiment, the four types of genes described above are introduced into cells having the ability to differentiate into oocytes such as pluripotent stem cells, and the cells are incubated for 5 to 10 days. Differentiation into immature oocytes can be induced by culturing for a short period of time as follows. Furthermore, in the case of humans, it takes 9 months or more to induce differentiation from primordial germ cells to immature oocytes in vivo. The period can be shortened dramatically.

Moreover, in the conventional method, it was necessary to go through at least two steps with different culture conditions in order to induce the differentiation of pluripotent stem cells into PGCLCs and further induce the differentiation of the PGCLCs into immature oocytes. In contrast, in the method for inducing immature oocytes of the present embodiment, pluripotent stem cells can be directly induced to differentiate into immature oocytes.

As used herein, the term "immature oocyte" refers to a primary oocyte that has not undergone follicular growth. An immature oocyte does not necessarily have to adopt a follicular structure. In immature oocytes, some maternal effect genes such as the Stella gene and the Padi6 gene, which are oocyte markers, are expressed as shown in Examples described later.
In the present specification, the term "follicle" is composed of an oocyte and somatic cells (granulosa cells and capsule cells) surrounding it.
The method for inducing immature oocytes of this embodiment will be described in detail below.

[Oocyte formation gene]
Oocyte formation genes used for introduction into cells that have the potential to differentiate into oocytes such as pluripotent stem cells have been studied by the inventors by RNA sequencing (RNA -Seq) was identified by performing analysis. Specific examples of oocyte formation genes include FIGLA, NOBOX, SOHLH1, LHX8, SUB1, STAT3, TBPL2, and DYNLL1. As shown in the examples below, four genes consisting of FIGLA, NOBOX, LHX8 and TBPL2 are particularly important for the induction of immature oocytes from pluripotent stem cells. Therefore, among the above-described oocyte-forming genes, at least four genes consisting of FIGLA, NOBOX, LHX8, and TBPL2, or their transcripts or expressed proteins, can be introduced into cells to enhance the ability to differentiate into oocytes. Cells with cytoplasm can be induced into immature oocytes.

In addition to the four types of genes or their transcripts or expressed proteins, it is preferable to further introduce the STAT3 gene, or its transcripts or expressed proteins, and it is more preferable to further introduce the STAT3 gene. As shown in Examples described later, by introducing STAT3 into cells in addition to FIGLA, NOBOX, LHX8 and TBPL2, the oocyte formation rate can be further improved.

Further, in addition to five types of genes, FIGLA, NOBOX, LHX8, TBPL2 and STAT3, or their transcripts or expressed proteins, one or more genes selected from the group consisting of SOHLH1, SUB1 and DYNLL1, or their It is preferable to further introduce transcripts or expressed proteins, more preferably three types of genes consisting of SOHLH1, SUB1 and DYNLL1, or their transcripts or expressed proteins. As shown in the examples below, by introducing all of the above eight types of genes, or their transcripts or expressed proteins into cells, cells capable of differentiating into oocytes are more efficiently transformed into immature oocytes. can be guided well.

It should be noted that the FIGLA gene is a basic helix-loop-helix (bHLH ) encode transcription factors. The transcription factor FIGLA binds to the E-box (5'-CANNTG-3') of the ZP (ZP1, ZP2 and ZP3) promoters. Diseases associated with FIGLA include, for example, premature ovarian failure (type 6), pseudohermaphroditism, and the like. Gene Ontology (GO) annotations of FIGLA include sequence-specific DNA binding and protein dimerization activities. An important paralog of FIGLA is SCX. FIGLA stands for Folliculogenesis Specific BHLH Transcription Factor, Factor In The Germline Alpha, Folliculogenesis-Specific Basic Helix-Loop-Helix Protein, Transcription F actor FIGa, BHLHC8 (BHLHc8), Folliculogenesis Specific Basic Helix-Loop-Helix, FIGALPHA (FIGalpha), POF6 Also called

Information on the base sequences of oocyte formation genes such as the FIGLA gene, the base sequences of the mRNAs of the genes, and the amino acid sequences of the proteins encoded by the genes can be obtained from databases such as Genbank.

The base sequence of the human FIGLA gene is disclosed, for example, in Genbank as "Gene ID: 344018". The nucleotide sequence of the mRNA of the human FIGLA gene is disclosed, for example, as Genbank accession number NM_001004311. The amino acid sequence of human FIGLA is disclosed in Genbank under accession number NP_001004311.

The nucleotide sequence of the mouse FIGLA gene is disclosed, for example, in Genbank as "Gene ID: 26910". The nucleotide sequence of the mRNA of the mouse FIGLA gene is disclosed, for example, as Genbank Accession No. NM — 012013. The amino acid sequence of mouse FIGLA is disclosed in Genbank under accession number NP_036143.

NOBOX genes encode transcription factors involved in oogenesis. Diseases associated with NOBOX include, for example, premature ovarian failure (type 5). NOBOX GO annotations include DNA binding transcription factor activity and RNA polymerase II near-promoter sequence-specific DNA binding. Specifically, it preferably binds to nucleotide sequences such as "5'-TAATTG-3'", "5'-TAGTTG-3'" and "5'-TAATTA-3'". An important paralog of NOBOX is UNCX. NOBOX is NOBOX Oogenesis Homeobox, Homeobox Protein NOBOX, Newborn Ovary Homeobox-Encoding Gene, Newborn Ovary Homeobox-Encoding, TCAG_12042, OG-2 (OG 2), also called OG2X, POF5.

The base sequence of the human NOBOX gene is disclosed, for example, in Genbank as "Gene ID: 135935". The nucleotide sequence of the mRNA of the human NOBOX gene is disclosed, for example, under Genbank accession numbers NM_001080413 and XM_001134420. The amino acid sequence of human NOBOX is disclosed in Genbank under accession numbers NP_001073882, XP_001134420.

The nucleotide sequence of the mouse NOBOX gene is disclosed, for example, as “Gene ID: 18291” in Genbank. The nucleotide sequence of the mRNA of the mouse NOBOX gene is disclosed, for example, as Genbank accession number NM_130869. The amino acid sequence of mouse NOBOX is disclosed in Genbank under accession number NP_570939.

The SOHLH1 gene is one of the gonad-specific transcription factors essential for spermatogenesis, oogenesis and folliculogenesis, and encodes a basic helix-loop-helix (bHLH) transcription factor. SOHLH1 has a role in regulating oocyte differentiation without affecting meiosis I. Diseases associated with SOHLH1 include, for example, non-obstructive azoospermia and ovarian hypoplasia. GO annotations of SOHLH1 include DNA-binding transcription factor activity and protein dimerization activity. An important paralog of the SOHLH1 gene is SOHLH2. SOHLH1 is Spermatogenesis And Oogenesis Specific Basic Helix-Loop-Helix 1, Spermatogenesis-And Oogenesis-Specific Basic Helix-Loop-Helix-Containing Protein 1, Spermatogenesis Associated 27, C9orf157, NOHLH, TEB2, Chromosome 9 Open Reading Frame 157, Newborn Also called Ovary Helix Loop Helix, BA100C15.3, SPATA27, BHLHe80, SPGF32, ODG5.

The nucleotide sequence of the human SOHLH1 gene is disclosed, for example, in Genbank as “Gene ID: 402381”. The nucleotide sequence of the mRNA of the human SOHLH1 gene is disclosed, for example, under Genbank accession numbers NM_001012415 and XM_497082. The amino acid sequence of human SOHLH1 is disclosed under Genbank accession numbers NP_001012415, XP_497082.

The nucleotide sequence of the mouse SOHLH1 gene is disclosed, for example, in Genbank as "Gene ID: 227631", and the nucleotide sequence of the mRNA of the mouse SOHLH1 gene is disclosed, for example, in Genbank under accession numbers NM_001001714 and XM_130180. The amino acid sequence of mouse SOHLH1 is disclosed in Genbank under accession numbers NP_001001714, XP_130180.

LHX8 is a member of the LIM homeobox family of proteins and is involved in patterning and differentiation of various tissues. The LIM homeobox family of proteins contains, in addition to the DNA-binding homeodomain, two tandemly repeated cysteine-rich double zinc finger motifs known as LIM domains. The LIM homeobox family of proteins are transcription factors involved in tooth morphogenesis, oogenesis and neuronal differentiation. Diseases associated with the LHX8 gene include, for example, cleft palate and odontoma. LHX8 is also called LIM Homeobox 8, LIM/Homeobox Protein Lhx8, LIM-Homeodomain Protein Lhx8, LIM Homeobox Protein 8, and LHX7.

The nucleotide sequence of the human LHX8 gene is disclosed, for example, in Genbank as “Gene ID: 431707”. The nucleotide sequence of the mRNA of the human LHX8 gene is disclosed, for example, under Genbank accession numbers NM_001001933, XM_086344, NM_001256114, XM_017001316, and XM_017001317. The amino acid sequence of human LHX8 is disclosed in Genbank under accession numbers NP_001001933, XP_086344, NP_001243043, XP_016856805, XP_016856806.

The nucleotide sequence of the mouse LHX8 gene is disclosed, for example, as "Gene ID: 16875" in Genbank. The nucleotide sequence of the mRNA of the mouse LHX8 gene is disclosed, for example, under Genbank accession numbers NM_010713, XM_006501072, and XM_017319470. The amino acid sequence of mouse LHX8 is disclosed in Genbank under accession numbers NP_034843, XP_006501135, XP_017174959.

The SUB1 gene is a gene that encodes a transcriptional regulator. SUB1 functions in concert with TAFs and acts as a coactivator that mediates functional interactions between upstream activators and general transcriptional functions. Diseases associated with SUB1 include, for example, tinea unguium. The GO annotation of SUB1 includes single-stranded DNA binding. SUB1 is SUB1 Homolog, Transcriptional Regulator, Positive Cofactor 4, Activated RNA Polymerase II Transcriptional Coactivator P15, PC4, P14, Activated RNA Polymerase II Also called Transcription Cofactor 4, RPO2TC1, P15.

The nucleotide sequence of the human SUB1 gene is disclosed, for example, in Genbank under “Gene ID: 10923”. The nucleotide sequence of the mRNA of the human SUB1 gene is disclosed, for example, as Genbank accession numbers NM_006713, XM_017008986, XM_017008987, and XM_011513944. The amino acid sequence of human SUB1 is disclosed in Genbank under accession numbers NP_006704, XP_016864475, XP_016864476, XP_011512246.

The nucleotide sequence of the mouse SUB1 gene is disclosed, for example, as “Gene ID: 20024” in Genbank. The nucleotide sequence of the mRNA of the mouse SUB1 gene is disclosed, for example, under Genbank accession numbers NM_011294 and XM_006520042. The amino acid sequence of mouse SUB1 is disclosed in Genbank under accession numbers NP_035424, XP_006520105.

STAT3 is a STAT protein family member. The STAT protein family includes interferon (IFN), epidermal growth factor (EGF), interleukin 5 (IL5), interleukin 6 (IL6), hepatocyte growth factor (HGF), leukemia inhibitory factor (LIF), bone morphogenetic protein 2 In response to cytokines and growth factors such as (BMP2), they are phosphorylated by receptor-associated kinases, then form homo- or heterodimers, which translocate to the cell nucleus where they act as transcriptional activators, leading to cell proliferation and apoptosis. It plays an important role in many cellular processes such as Diseases associated with STAT3 include, for example, childhood-onset multisystemic autoimmune diseases, autosomal dominant hyper-IgE syndrome, and the like. GO annotations of STAT3 include DNA-binding transcription factor activity and sequence-specific DNA-binding. An important paralog of the STAT3 gene is STAT1. STAT3 is Signal Transducer And Activator Of Transcription3, Acute-Phase Response Factor, APRF, Signal Transducer And Activator Of Transcription 3, DNA-Binding Protein AP, Also called ADMIO1, ADMIO, HIES.

The nucleotide sequence of the human STAT3 gene is disclosed, for example, as "Gene ID: 6774" in Genbank. The base sequences of the mRNAs of the human STAT3 gene are, for example, Genbank Accession Nos. 369520, NM_003150, NM_139276, NM_213662, XM_017024973, XM_011525146, XM_011525145, XM_017024972, XM_005257617, XM_005257616 , XM_017024975, XM_024450896, XM_017024974, XM_017024976. The amino acid sequence of human STAT3 is Genbank Accession Nos. NP_003141, NP_644805, NP_998827, XP_016880462, XP_011523448, XP_011523447, XP_016880461, XP_005257674, XP_005257673, XP_016880464, XP _024306664, XP_016880463 , XP_016880465.

The nucleotide sequence of the mouse STAT3 gene is disclosed, for example, in Genbank under “Gene ID: 20848”. The nucleotide sequences of the mRNAs of the mouse STAT3 gene are disclosed, for example, under Genbank accession numbers NM_011486, NM_213659, NM_213660, XM_011248846, and XM_017314401. The amino acid sequence of mouse STAT3 is disclosed in Genbank under accession numbers NP_035616, NP_998824, NP_998825, XP_011247148, XP_017169890.

The TBPL2 gene is a gene that encodes TATA-box binding protein-like 2. TBPL2 is a transcription factor that forms a complex with TAF3 to induce differentiation of myoblasts into muscle cells, and the complex displaces TFIID in specific promoters early in differentiation. Diseases associated with TBPL2 include, for example, retinitis pigmentosa. The GO annotation of TBPL2 includes a DNA-binding transcription factor activity. An important paralog of the TBPL2 gene is TBP. TBPL2 is TATA-Box Binding Protein-Like 2, TATA Box-Binding Protein-Related Factor 3, TATA Box-Binding Protein-Like Protein 2, TBP-Related Factor 3, TBP-Like Protein 2, TBP2 , also called TRF3.

The nucleotide sequence of the human TBPL2 gene is disclosed, for example, as "Gene ID: 387332" in Genbank. The nucleotide sequence of the mRNA of the human TBPL2 gene is disclosed, for example, as Genbank Accession No. NM_199047. The amino acid sequence of human TBPL2 is disclosed in Genbank under accession number NP_950248.

The nucleotide sequence of the mouse TBPL2 gene is disclosed, for example, in Genbank as “Gene ID: 227606”. The nucleotide sequence of the mRNA of the mouse TBPL2 gene is disclosed, for example, under Genbank accession numbers NM_001289689 and NM_199059. The amino acid sequence of mouse TBPL2 is disclosed under Genbank accession numbers NP_001276618, NP_951014.

The DYNLL1 gene is a gene that encodes a protein classified as a light chain among proteins that constitute cytoplasmic dynein, which is an enzyme complex with a molecular weight of about 1200 kDa. Diseases associated with DYNLL1 include, for example, chronic intestinal venous insufficiency. GO annotations of DYNLL1 include protein homodimerization activity and protein domain-specific binding. An important paralog of the DYNLL1 gene is DYNLL2. DYNLL1 is Dynein Light Chain LC8-Type 1, Protein Inhibitor Of Neuronal Nitric Oxide Synthase, Dynein, Cytoplasmic, Light Polypeptide 1, Dynein Light C hain 1, Cytoplasmic, 8 KDa Dynein Light Chain, DNCLC1, DNCL1, DLC1, DLC8, PIN, Also called Cytoplasmic Dynein Light Polypeptide, Hdlc1 (HDLC1), LC8a, LC8.

The nucleotide sequence of the human DYNLL1 gene is disclosed, for example, as "Gene ID: 8655" in Genbank. The nucleotide sequence of the mRNA of the human DYNLL1 gene is disclosed, for example, under Genbank accession numbers NM_001037494, NM_001037495, and NM_003746. The amino acid sequence of human DYNLL1 is disclosed in Genbank under accession numbers NP_001032583, NP_001032584, NP_003737.

The nucleotide sequence of the mouse DYNLL1 gene is disclosed, for example, as “Gene ID: 56455” in Genbank. The nucleotide sequence of the mRNA of the mouse DYNLL1 gene is disclosed, for example, as Genbank Accession No. NM_019682. The amino acid sequence of mouse DYNLL1 is disclosed in Genbank under accession number NP_062656.

[Cells capable of differentiating into oocytes]
Cells capable of differentiating into oocytes used in the method for inducing immature oocytes of the present embodiment are selected from the group consisting of pluripotent stem cells, epiblast-like cells (EpiLCs) and primordial germ cells. At least one type of cell is preferred.

(pluripotent stem cells)
As used herein, the term "pluripotent stem cell" means an undifferentiated cell having "self-renewal ability" that can proliferate while maintaining an undifferentiated state and "pluripotency" that can differentiate into all three germ layer lineages. do. Examples of pluripotent stem cells include, but are not limited to, induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic germ cells derived from primordial germ cells (EG cells), and testis tissue. multipotent GS cells (mGS cells) isolated in the process of establishing GS (Germline Stem) cells from cereals, Muse cells isolated from bone marrow mesenchymal cells, and the like. The ES cells may be ES cells generated by nuclear reprogramming from somatic cells. The pluripotent stem cells listed above can be obtained by known methods.

As used herein, the term “iPS cells” means cells that have become capable of being reprogrammed into cells of various tissues and organs by introducing several genes into differentiated somatic cells. The iPS cells used in the method for inducing immature oocytes of the present embodiment may be derived from primary cultured somatic cells collected from a suitable donor, or may be derived from an established cell line. Since iPS cells can be induced to differentiate into any germ layer cells, the somatic cells used for the preparation of iPS cells can in principle be derived from either ectodermal or endoderm cells. There may be. Skin cells, hair cells, gingiva cells, blood cells, etc., which are less invasive and can be easily collected, are suitable as somatic cells used for the preparation of iPS cells. A method for preparing iPS cells may be according to a method known in the art. Specifically, for example, “Okita K. et al., “Generation of germline-competent induced pluripotent stem cells.”, Nature, Vol. 448, p313-317, 2007.” (Reference 1), “Hamanaka S et al., “Generation of germline-competent rat induced pluripotent stem cells.”, PLoS One, Vol. 6, Issue 7, e22008, 2011.” (Reference 2). method available.

ES cells used in the method for inducing immature oocytes of the present embodiment can be obtained by known methods. For example, it can be established by collecting an inner cell mass from a blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on fibroblast-derived feeder cells. In addition, ES cells established by culturing early embryos produced by nuclear transfer of somatic cell nuclei can also be used. In addition, as shown in Examples described later, ES cells can be maintained and cultured in a serum-free medium supplemented with 2i (2 inhibitors; PD0325901 and CHIR99021) and LIF (Leukemia Inhibitory Factor) without using feeder cells. (Reference 3: “Ying QL et al., “The ground state of embryonic stem cell self-renewal.”, Nature, Vol. 453, No. 7194, p519-523, 2008.”).

(epiblast-like cells)
As used herein, "epiblast-like cells (EpiLCs)" are cells differentiated from pluripotent stem cells (e.g., iPS cells, ES cells, etc.) under specific culture conditions, and epiblasts (in vivo tissue that differentiates into primordial germ cells at
Methods for inducing differentiation of EpiLCs from pluripotent stem cells (iPS cells or ES cells) are known methods, for example, Japanese Patent Publication No. 2013-538038 (reference document 4), "Hayashi K. et al., "Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells.”, Cell, Vol. 146, No. 4, p519-532, 2011.” (reference document 5).

(primordial germ cell)
As used herein, the term “primordial germ cell” refers to a cell that is scheduled to differentiate into a germ cell, and means a cell that undergoes meiosis and ultimately differentiates into an ovum or sperm. The primordial germ cells may be derived from a living organism, or may be primordial germ cell-like cells (PGCLCs) differentiated from pluripotent stem cells. When primordial germ cells are collected from a living organism, they can be collected together with gonads from fetuses of female mice (11.5 to 12.5 days old), for example. When the gonad is collected from the living body, it may be collected together with the mesonephros, or may be collected by separating the mesonephros.

In addition, as described above, "primitive germ cells" include primordial germ cell-like cells differentiated from pluripotent stem cells. Methods for inducing differentiation of PGCLCs from pluripotent stem cells (iPS cells or ES cells) can be performed by referring to known methods, for example, reference 5 above.

When PGCLCs derived from pluripotent stem cells are used as primordial germ cells, it is preferable to previously remove undifferentiated cells from the differentiation-induced pluripotent stem cell population. Such a method is known. For example, by introducing a nucleic acid encoding a fusion protein in which Blimp1, a primordial germ cell marker gene, and a reporter protein are bound to pluripotent stem cells, pluripotent stem cells can be obtained. PGCLCs induced to differentiate from stem cells and undifferentiated cells can be easily separated by a fluorescence-activated cell sorting (FACS) method or the like.

In addition, "primordial germ cells" also include cells in which the genes of living body-derived primordial germ cells and pluripotent stem cell-derived primordial germ cell-like cells are modified using genetic engineering techniques. Methods for modifying the genes of living body-derived primordial germ cells and pluripotent stem cell-derived primordial germ cell-like cells include the CRISPR system, a method using Transcription Activator-Like Effector Nucleases (TALEN), and a method using zinc finger nuclease. A target nucleic acid, vector, or the like can be introduced using a known genome editing method such as a homologous recombination method. Methods for introducing nucleic acids, vectors, and the like include, for example, microinjection, electroporation, lipofection, nucleic acid introduction using viral vectors, and the like. In addition, the method for introducing a foreign gene or foreign nucleic acid fragment is not limited to the methods listed above as long as genetically modified primordial germ cells can be differentiated into functional oocytes by the method of the present embodiment. Genetic modification of primordial germ cells can be performed at an appropriate timing during the culture period of primordial germ cells. For example, in mice, it can be performed between 11.5 and 12.5 days of age. Moreover, when using primordial germ cell-like cells derived from pluripotent stem cells, the pluripotent stem cells before induction of differentiation into primordial germ cell-like cells can be genetically modified by a known method.

Among them, pluripotent stem cells are preferable as cells having the ability to differentiate into oocytes used in the method for inducing immature oocytes of the present embodiment. In particular, when ES cells are used, a large amount of immature oocytes can be obtained due to their high proliferation ability.

In addition, mammal-derived cells can be used as cells having the ability to differentiate into oocytes. Mammals include, but are not limited to, humans, chimpanzees and other primates; dogs, cats, rabbits, horses, sheep, goats, cows, pigs, rats (including nude rats), mice (nude mice and skid mice), livestock animals such as hamsters and guinea pigs, pet animals and laboratory animals, etc., but are not limited thereto.

[Introduction process]
In the method for inducing immature oocytes of this embodiment, the oocyte formation gene is introduced into at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells.

Also, instead of the oocyte formation gene, its transcript, mRNA, or its expressed protein may be introduced. As the mRNA of the oocyte-forming gene and its expressed protein, the mRNA consisting of the nucleotide sequence indicated by the accession number in Genbank and the expressed protein consisting of the amino acid sequence can be used.

The introduction method is not particularly limited, and can be appropriately selected according to the target cell and the type of material to be introduced (nucleic acid, protein, etc.).

The method for introducing the oocyte-forming gene into cells is not particularly limited, and a known method can be appropriately selected and used. Specific examples include the lipofection method, the microinjection method, the DEAE dextran method, the gene gun method, the electroporation method, the calcium phosphate method and the like.

The method for introducing the mRNA of the oocyte-forming gene into cells is not particularly limited, and a known method can be appropriately selected and used. Specifically, for example, a method using a commercially available RNA transfection reagent such as Lipofectamine (registered trademark) MessengerMAX (manufactured by Life Technologies) can be used.

The method for introducing the expressed protein of the oocyte-forming gene into cells is not particularly limited, and a known method can be appropriately selected and used. Specific examples include a method using a protein transfer reagent, a method using a protein transfer domain (PTD) fusion protein, a microinjection method, and the like.

The oocyte-forming gene may be introduced in the form of an expression vector into at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells, and transiently expressed. The cytogenic gene may be integrated into the chromosome of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells. Among them, it is preferable to integrate the oocyte formation gene into the chromosome of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells, since the gene can be stably expressed.

When the oocyte-forming gene is introduced in the form of an expression vector into at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells, the base sequence of the oocyte-forming gene; An expression vector containing a promoter that controls the expression of the nucleotide sequence of the oocyte formation gene can be used. In the expression vector, the nucleotide sequence of the oocyte formation gene is operably linked to a promoter. In addition, among the above eight oocyte formation genes, all of the genes to be used may be incorporated into one expression vector, or they may be incorporated into different vectors one by one. It is preferable to integrate all the genes to be used into one expression vector.

The promoter is not particularly limited, and may be one having activity in target cells, or may be an expression-inducible promoter whose activity can be induced by a drug or the like.

Promoters active in target cells include, for example, cytomegalovirus promoter (CMV promoter), CMV early enhancer/chicken beta actin (CAG promoter), etc., which have strong promoter activity in almost all cells.

Examples of expression-inducible promoters include doxycycline-inducible promoters (TetO promoters) whose promoter activity can be artificially controlled.

The expression vector contains, in addition to the base sequence and promoter of the oocyte-forming gene, if desired, an enhancer, a poly(A) addition signal, a marker gene, a replication origin, and a gene encoding a protein that binds to the replication origin to control replication. etc. may be included. A "marker gene" refers to a gene that enables sorting and selection of cells by introducing the marker gene into cells. Specific examples of marker genes include drug resistance genes, fluorescent protein genes, luciferase genes, chromogenic enzyme genes, and the like. These may be used individually by 1 type, and may use 2 or more types together. Specific examples of the drug resistance gene include puromycin resistance gene, geneticin resistance gene, neomycin resistance gene, tetracycline resistance gene, kanamycin resistance gene, zeocin resistance gene, hygromycin resistance gene, chloramphenicol resistance gene, and the like. mentioned. Specific examples of the fluorescent protein gene include green fluorescent protein (GFP) gene, yellow fluorescent protein (YFP) gene, red fluorescent protein (RFP) gene, and the like. Specific examples of the luciferase gene include, for example, a luciferase gene. Specific examples of the chromogenic enzyme gene include β-galactosidase gene, β-glucuronidase gene, alkaline phosphatase gene and the like.

The expression vector into which the oocyte formation gene is incorporated is not particularly limited, and known expression vectors can be used. Expression vectors include, for example, plasmid vectors, virus vectors and the like.

The plasmid vector is not particularly limited as long as it can be expressed in at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells. For example, commonly used mammalian cell expression plasmid vectors can be used. Examples of mammalian cell expression plasmid vectors include, but are not limited to, pX459, pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo and the like.

Viral vectors include, for example, retrovirus (including lentivirus) vectors, adenovirus vectors, adeno-associated virus vectors, Sendai virus vectors, herpes virus vectors, vaccinia virus vectors, pox virus vectors, polio virus vectors, silbis virus vectors , rhabdovirus vector, paramyxovirus vector, orthomyxovirus vector and the like.

Among them, a plasmid vector is preferable as the expression vector.

A known knock-in system can be used to integrate the oocyte formation gene into the chromosome. Known knock-in systems include, for example, CRISPR/Cas system, Transcription Activator-Like Effector Nucleases (TALEN), and zinc finger nuclease. Examples include a method of homologous recombination, a method using a transposon vector system, and the like.

The donor vector contains base sequences that flank the target region as homology arms. The donor vector can contain the nucleotide sequence of the oocyte formation gene (hereinafter sometimes referred to as "knock-in sequence") between the 5' arm and the 3' arm. Moreover, in order to stably express the oocyte formation gene, it is preferable to set the target region within the safe harbor region.

Donor vectors may be circular DNA vectors (eg, plasmid vectors) or linear DNA vectors. Donor vectors may contain other sequences in addition to homology arms and knock-in sequences. Other sequences include, for example, marker genes, origins of replication, genes encoding proteins that bind to origins of replication to control replication, and the like. Marker genes include the same ones as described above.

A method for introducing the donor vector is not particularly limited, and can be appropriately selected according to the target cell. Methods for introducing a donor vector into cells include, for example, the lipofection method, the microinjection method, the DEAE dextran method, the gene gun method, the electroporation method, the calcium phosphate method and the like.

In the transposon vector system, as shown in Examples described later, a transposon vector in which the base sequence of the oocyte-forming gene is integrated is introduced into cells, and transposase is allowed to act to facilitate integration into the chromosome of the cells. can be done. Further, by allowing the transposase to act again, the nucleotide sequence of the above-mentioned oocyte formation gene integrated into the chromosome can be excised from the chromosome and removed without leaving a trace. Examples of transposons include piggyBac (registered trademark), Sleeping Beauty, Tol II, mariner, and the like.

The method for inducing immature oocytes of the present embodiment may include any step in addition to the introduction step. Optional steps include, for example, a step of proliferating cells (proliferation step), a step of selecting cells into which the introduced oocyte formation gene, or its transcript or its expression protein has been introduced (selection step), Examples thereof include a step of culturing the cells after the introduction step in a state in which the oocyte-forming gene is expressed or the expressed protein of the oocyte-forming gene is present in the cells (culturing step). Moreover, in the introducing step, when an oocyte-forming gene is introduced, a step of inducing the expression of the introduced oocyte-forming gene (expression-inducing step) can be included.

[Proliferation step]
In the proliferation step, at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells is proliferated in order to obtain a larger amount of immature oocytes.

In the growth step, for example, at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells can be grown by culturing it in a growth medium. As the growth medium, known media for culturing ES cells, iPS cells, EpiLCs, primordial germ cells, etc. can be used, but not limited thereto, ES cells, iPS cells, EpiLCs, primordial germ cells. Any medium may be used as long as it is suitable for culturing of Specific examples of growth media include serum-free media supplemented with 2i (2 inhibitors; PD0325901 and CHIR99021) and LIF (Leukemia Inhibitory Factor), as shown in Examples below.

As culture conditions in the proliferation step, known conditions for culturing at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells can be used. Specifically, for example, the culture temperature can be about 30° C. or higher and 37° C. or lower. The culture period for mice is not particularly limited, but can be, for example, about 1 to 10 days, or about 3 to 7 days. Moreover, a person skilled in the art can appropriately set a preferable culture period depending on the derived animal species.

The proliferation step may be performed before or after the introduction step. In the case after the introduction step, if the oocyte-forming gene is expressed or the expression protein of the oocyte-forming gene is present, it consists of pluripotent stem cells, EpiLCs, and primordial germ cells. At least one type of cell selected from the group initiates differentiation induction into immature oocytes, thereby stopping proliferation. Therefore, when the expression of the oocyte-forming gene is controlled to be induced by the presence of an expression-inducing substance, as shown in the expression-inducing step described later, after the introduction step, the absence of the expression-inducing substance A growth step can be performed below.

In addition, when introducing an oocyte-forming gene in the form of a transient expression vector, or when introducing a transcript or expression protein of an oocyte-forming gene, a growth step is carried out before the above-mentioned introduction step. preferably done.

[Selection process]
In the selection step, cells into which the oocyte formation gene, or transcript or expressed protein thereof has been introduced are selected.

In the selection step, cells into which the oocyte formation gene, or its transcript or its expressed protein has been introduced can be selected, for example, by using a reporter gene. Specifically, for example, when an oocyte-forming gene is introduced into a cell in the form of an expression vector, the inclusion of a reporter gene in the expression vector results in the expression of the oocyte-forming gene in the cell, Alternatively, cells can be selected by expressing the reporter gene independently of the expression of the oocyte formation gene. When the oocyte formation gene is integrated into the chromosome of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells, a reporter gene is functionally placed upstream or downstream of the oocyte formation gene. By incorporating a construct linked to , cells can be selected for expression of the reporter gene with or independently of the expression of the oocyte-forming gene. When a transcript of an oocyte-forming gene is introduced, a construct in which a transcript of a reporter gene is functionally linked upstream or downstream of the transcript of the oocyte-forming gene is introduced into a cell, thereby producing an egg. Cells can be selected by expressing a reporter gene in conjunction with the expression of the mothering gene. When an expressed protein of an oocyte-forming gene is introduced into a cell, cells can be selected by introducing a fusion protein of an expressed protein of the oocyte-forming gene and an expressed protein of a reporter gene into the cell. . As the reporter gene, those exemplified as the marker gene in the explanation of the "introduction step" above can be used.

[Expression induction step]
In the introduction step, when the expression of the oocyte-forming gene is controlled to be induced by the presence of an expression-inducing substance, the expression of the oocyte-forming gene is induced by adding the expression-inducing substance to the medium. Induced. When at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells is induced to differentiate into immature oocytes, cell proliferation stops. Therefore, in order to obtain a larger amount of immature oocytes, it is preferable to carry out the expansion step before carrying out the expression induction step. That is, when an oocyte-forming gene is introduced into cells in the form of an expression vector, it is preferable to carry out the steps of proliferation, introduction and induction of expression in this order. On the other hand, when the oocyte formation gene is integrated into the chromosome of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells, the introduction step, proliferation step and expression induction step are performed in this order. preferably done.

Expression of the oocyte-forming gene is induced by, for example, introducing the oocyte-forming gene into cells in a form operably linked to an expression-inducible promoter (e.g., doxycycline-inducible promoter (TetO promoter)), and inducing expression. A method of expressing an oocyte-forming gene by adding a substance (eg, doxycycline) to the medium, and the like. Alternatively, for example, as shown in Examples described later, a method using the ProteoTuner (registered trademark) system (manufactured by Clontech) can be used. Specifically, by introducing into cells a construct in which a sequence encoding a destabilizing domain (DD, 12 kDa) is functionally linked upstream or downstream of an oocyte formation gene, the construct is The expressed fusion protein is rapidly degraded by the proteasome in the absence of an expression inducer. On the other hand, by adding a low-molecular-weight compound Shield1 (membrane-permeable low-molecular-weight compound, 750 Da) to the medium as an expression inducer, which protects against degradation by the proteasome, the oocyte-forming gene is stably expressed and intracellularly. can be accumulated.

The amount of the expression-inducing substance to be added is not particularly limited as long as the concentration is such that the expression level of the oocyte-forming gene is the desired level. For example, when the expression inducer is doxycycline, the concentration in the medium can be, for example, about 1 nM or more and 10 μM or less. When the expression inducer is Shield1, the concentration in the medium can be, for example, about 10 nM or more and 10 μM or less.

As the medium used in the expression induction step, those exemplified as growth media in the growth step can be used.

[Culturing process]
In the culturing step, the cells after the introduction step are cultured in a state in which the oocyte-forming gene is expressed or the expressed protein of the oocyte-forming gene is present in the cells.

As culture conditions, known conditions for culturing at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells can be used. Specifically, for example, the culture temperature can be about 30° C. or higher and 37° C. or lower. In the case of mice, the culture period can be about 1 to 10 days, and can be about 3 to 7 days.

As the medium used in the culture step, those exemplified as growth media in the growth step can be used. In addition, when the expression of the oocyte-forming gene is controlled to be induced by the presence of an expression-inducing substance, the expression-inducing substance is added to the medium, and the oocyte-forming gene is expressed in the cells. Cultivate in a state where

A preferred embodiment of the method for inducing immature oocytes includes the following 1) to 5), wherein the expression of the oocyte-forming gene is controlled to be induced by the presence of an expression-inducing substance.
1) at least one oocyte formation gene selected from the group consisting of FIGLA, NOBOX, SOHLH1, LHX8, SUB1, STAT3, TBPL2 and DYNLL1 selected from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells introduction into the chromosome of one type of cell (particularly preferably, pluripotent stem cells);
2) growing the cells after introduction;
3) Selecting cells in which the oocyte formation gene has been introduced into the chromosome from the cells after proliferation;
4) adding an expression inducer to the medium to induce expression of the oocyte-forming gene in the cells after selection;
5) culturing the cells after expression induction in a state in which the oocyte formation gene is expressed in the cells

In the method for inducing immature oocytes of the present embodiment, as described above, a short culture period of about 5 days or more and 10 days or less after the introduction of the oocyte-forming gene, its transcript, or its expression protein is performed. At least one type of cell selected from the group consisting of potential stem cells, EpiLCs and primordial germ cells can be induced to differentiate into immature oocytes.

The fact that at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells was induced to differentiate into immature oocytes was confirmed by a known oocyte marker gene (e.g., Stella, etc.). It can be confirmed from the expression. Specifically, as shown in Examples described later, a fusion protein in which Stella, an oocyte marker gene, and a reporter protein (for example, enhanced cyan fluorescent protein (ECFP)) are bound together. By introducing a nucleic acid encoding in advance into the chromosome of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells, the fluorescence detected by the expression of Stella-ECFP shows immature It can be confirmed that the cells were induced to differentiate into oocytes. Furthermore, from the expression of Stella-ECFP, undifferentiated cells can be removed from the differentiation-induced cell population by fluorescence-activated cell sorting (FACS) or the like, and immature oocytes can be easily separated. .

<Method for preparing mature oocytes>
In one embodiment, the present invention provides one or more genes selected from the group consisting of FIGLA, NOBOX, SOHLH1, LHX8, SUB1, STAT3, TBPL2 and DYNLL1, or transcripts or expressed proteins thereof, in pluripotent stem cells, EpiLCs. and primordial germ cells, and co-culturing the introduced cells and ovarian somatic cells. .

In the method for producing mature oocytes of the present embodiment, the introduction of the oocyte-forming gene (introduction step) is the same as the introduction step described in the above-mentioned "Method for Inducing Immature Oocytes", so explanation is given. Omit.

As used herein, the term "mature oocyte" means an egg in the second meiotic metaphase, and is also called a secondary oocyte. In addition, mature oocytes express the Npm2 gene, which is an activated oocyte marker, as shown in Examples described later.

[Agglomerate formation step]
The method for producing a mature oocyte of the present embodiment includes at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step described in the "Method for Inducing Oocytes" above. and co-cultivating ovarian somatic cells to form aggregates (aggregate formation step).

Ovarian somatic cells used in the aggregate formation step are somatic cells collected from living ovaries, and are co-cultured with at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells. Thus, they are cells that differentiate into granulosa cells and capsule cells that constitute follicles.

The method for collecting somatic cells from the ovary of a living body can be performed according to the method described in Reference Document 5 above.

Specifically, for example, in the method of collecting somatic cells from an ovary, the ovary is surgically collected from a living body, and the somatic cells constituting the ovary can be dissociated by trypsin treatment or the like. In addition, at this time, it is preferable to remove germ cells endogenous to the living body-derived ovary. A method for removing endogenous germ cells from the ovaries can be performed by a known method. For example, endogenous germ cells can be removed by magnetic activated cell sorting using an anti-SSEA1 antibody or an anti-CD31 antibody. Here, the ovary is preferably derived from a fetus in order to collect ovarian somatic cells. In the case of mice, for example, gonads (ovaries) derived from mouse embryos of 12.5 days of embryonic age (also referred to as “embryonic age”) can be used. Moreover, a person skilled in the art can appropriately select a gonad (ovary) at a suitable time according to the derived animal species based on the present disclosure and common technical knowledge in the relevant technical field.

In the aggregate formation step, by co-cultivating at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells after the introduction step, and ovarian somatic cells, pluripotency after the introduction step It is preferable to form an aggregate consisting of at least one cell selected from the group consisting of stem cells, EpiLCs and primordial germ cells, and ovarian somatic cells.

A method for producing an aggregate consisting of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells after the introduction step, and ovarian somatic cells is, for example, as shown in the examples below. , S10 medium (StemPro®-34 SFM, Life Technologies) supplemented with 10% fetal calf serum (FCS), 150 μM ascorbic acid, 1×Glutamax, 1× penicillin/streptomycin and 55 μM mercaptoethanol , at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells after the introduction step and ovarian somatic cells are mixed, aggregated and cultured. When the expression of the oocyte-forming gene is controlled to be induced by the presence of an expression-inducing substance, the expression-inducing substance is added to the medium to express the oocyte-forming gene. cultivated in a state It is preferable to use a low-adsorption culture dish (for example, low cell-adhesion U-bottom 96-well plate, etc.) for the culture.

In the case of mice, for example, the culture period for producing aggregates can be about 2 days or more and 3 days or less, preferably 2 days. Moreover, a person skilled in the art can appropriately set a preferable culture period depending on the derived animal species.

In addition, the ratio of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells after the introduction step and ovarian somatic cells when mixed is such that the produced aggregates form mature follicles. Although not limited as far as possible, for example, in the case of mice, the ratio of the cell number of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells after the introduction step to ovarian somatic cells is A ratio of about 2:1 is preferred.

In addition, at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells after the introduction step, ovarian somatic cells, or ovaries containing ovarian somatic cells may be cryopreserved. can. The cryopreservation method can be performed by a known method. For example, cryopreservation of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells after the introduction step and ovarian somatic cells can be performed in a 10% DMSO solution or a commercially available freezing agent (Cellbanker (registered trademark) ), etc.).

Note that the ovarian somatic cells used in the method for producing mature oocytes of the present embodiment are the same as at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells that constitute aggregates. It may be derived from one kind of mammal or from a different species of mammal, but it is preferable to use one derived from the same species of mammal. Examples of mammals include those exemplified in the above description of cells capable of differentiating into oocytes.

The method for producing mature oocytes of the present embodiment may include any step after the introduction step and before the aggregate formation step. Optional steps include the proliferation step, selection step, and the like described in the above-mentioned “method for inducing immature oocytes”.

[Culturing process]
In addition, the method for producing mature oocytes of the present embodiment can include a culture step after the aggregate formation step.

In the culturing step, it is preferable to transfer the aggregates after the aggregate formation step onto a collagen membrane and culture them.

As culture conditions, known conditions for culturing aggregates can be used. Specifically, for example, the culture temperature can be about 30° C. or higher and 37° C. or lower. In the case of mice, the culture period can be about 7 days or more and 35 days or less, and can be about 8 days or more and 30 days or less. Moreover, a person skilled in the art can appropriately set a preferable culture period depending on the derived animal species.

As the medium used in the culture step, those exemplified as growth media in the growth step can be used. In addition, when the expression of the oocyte-forming gene is controlled to be induced by the presence of an expression-inducing substance, the expression-inducing substance is added to the medium, and the oocyte-forming gene is expressed in the cells. Cultivate in a state where

Agglomerates after the culturing step have a secondary follicle structure, the oocyte is surrounded by multilayered granulosa cells, and further the theca folic surrounding the multilayered granulosa cells is formed. The theca cells that make up the theca interna of the follicle have a luteinizing hormone receptor, and the granulosa cells have a follicle stimulating hormone receptor. It is expressed.

Next, by culturing the follicles obtained in the culture step using the method described in Non-Patent Document 1, mature oocytes can be produced. The process of obtaining mature oocytes can be divided into a growth process and a maturation process.

[Growth process]
In the growth step, the follicles obtained in the culture step are isolated into individual follicles and cultured using a growth medium.

As culture conditions, the conditions described in Non-Patent Document 1 can be used. Specifically, for example, the culture temperature can be about 30° C. or higher and 37° C. or lower. In the case of mice, the culture period can be about 7 days or more and 15 days or less, and can be about 8 days or more and 13 days or less. Moreover, a person skilled in the art can appropriately set a preferable culture period depending on the derived animal species.

As a growth medium, a medium having the composition described in Non-Patent Document 1 can be used. Specifically, for example, in the case of mice, for two days from the start of culture, 5% fetal calf serum (FCS), 2% polyvinylpyrrolidone (manufactured by Sigma), 150 μM ascorbic acid, 1×GlutaMAX, 1× penicillin/streptomycin, 100 μM 2-mercaptoethanol, 55 μg/mL sodium pyruvate (Nacalai Tesque up to this point), 0.1 IU/mL follicle-stimulating hormone (Follistim (registered trademark), MSD), Follicles are cultured using α-MEM containing 15 ng/mL BMP15 (Bone morphogenetic protein 15) and 15 ng/mL GDF9 (Growth differentiation factor 9) (manufactured by R&D Systems). Then, on the second day from the start of the culture, the follicles are incubated in 0.1% Type IV collagenase (manufactured by MP Biomedicals), replaced with α-MEM from which BMP15 and GDF9 have been removed. Next, after washing several times with α-MEM containing 5% FCS, the follicles are cultured using α-MEM with the above composition except BMP15 and GDF9 from the start of culture until day 11.

The follicle after the growth process has an antral follicle structure and forms a cumulus-oocyte complex with the nucleus-vesicle-stage egg.

[Maturation process]
In the maturation step, the follicles obtained in the growth step are cultured using a maturation medium.

As culture conditions, the conditions described in Non-Patent Document 1 can be used. Specifically, for example, the culture temperature can be about 30° C. or higher and 37° C. or lower. In the case of mice, the culture period can be about 7 days or more and 15 days or less, and can be about 8 days or more and 13 days or less. Moreover, a person skilled in the art can appropriately set a preferable culture period depending on the derived animal species.

As a maturation medium, a medium having the composition described in Non-Patent Document 1 can be used. Specifically, for example, in the case of mice, 5% FCS, 25 μg/mL sodium pyruvate, 1× penicillin/streptomycin, 0.1 IU/mL follicle-stimulating hormone, 4 ng EGF (Epidermal Growth Factor), and α-MEM containing 1.2 IU/mL of hCG (human chorionic gonadotropin, abbreviation: gonadotropin, manufactured by ASKA).

The follicle after the maturation process has matured to the second meiotic metaphase egg (secondary oocyte).
Whether the egg is in the second meiotic metaphase (secondary oocyte) can be evaluated, for example, by visual observation using a microscope or the like, using the release of the first polar body as an index.

The obtained matured oocytes (secondary oocytes) are preferably used for infertility treatment. That is, in one embodiment, the present invention provides a method of treating infertility using mature oocytes obtained by the above method.
In addition, mature oocytes obtained using the method for producing mature oocytes of the present embodiment are suitably used for efficient breeding of industrial animals and breeding of rare animals. That is, in one embodiment, the present invention provides a method for breeding industrial animals or a method for breeding rare animals using mature oocytes obtained by the above method.
It should be noted that mammals are preferable as animals to which the above-mentioned application is applied. Mammals include those similar to those exemplified above.
In addition, mature oocytes obtained using the method for producing mature oocytes of the present embodiment can be used to investigate the causes of infertility and to elucidate the mechanisms of menopausal diseases.

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[Example 1]
(Vector construction)
The CAG promoter and destabilization domain (DD) were transferred to the CAG-DD-hTFAP2C plasmid (Reference 6: “Kobayashi T et al., “Principles of early human development and germ cell program from conserved model systems.”, Nature, Vol. 546, No. 7658, p416-420, 2017.”) and the conventionally used PiggyBAC vector (Reference 7: “Shimamoto S et al., “Hypoxia induces the dormant state in oocytes through expression of Foxo3 ”, PNAS, https://doi.org/10.1073/pnas.1817223116, 2019.”) to create a PB-CAG-DD vector. Next, the cDNAs of the eight genes of FIGLA, NOBOX, SOHLH1, LHX8, SUB1, STAT3, TBPL2 and DYNLL1 were amplified from the cDNA of 13.5-day-old female mouse ovary after fertilization, and the Infusion HD Cloning Kit (Takara Bio Inc.) was used. (manufactured) and cloned into the PB-CAG-DD vector. Amplification of each cDNA was performed by PCR using KOD Fx Neo or KOD Plus Neo DNA polymerase (manufactured by TOYOBO) according to the manufacturer's protocol.

(Vector transfection)
ES cells were maintained and cultured in advance in a serum-free medium supplemented with 2i and LIF without using feeder cells (see reference 3 above). In addition, to allow monitoring of differentiation into immature and mature oocytes, mVenus (membrane -targeted Venus), the gene encoding enhanced cyan fluorescent protein (ECFP) under the expression control of Stella, a germ cell and oocyte marker, and activation (maturation) Mouse ES cells (Blimp1-mVenus: Stella-ECFP: Blimp1-mVenus: Stella-ECFP: Npm2-mCherry (BVSCNmC)) was used. The constructed PB-CAG-DD vector containing the eight genes was co-transfected with the hyperactive PBase (hypBase) plasmid by Lipofectamine 2000. Single colonies were grown after puromycin selection for 5 days in serum-free medium supplemented with 2i and LIF.

(Differentiation induction into immature oocytes)
Then, S10 medium supplemented with 10% fetal calf serum (FCS), 150 μM ascorbic acid, 1×Glutamax, 1× penicillin/streptomycin and 55 μM mercaptoethanol (StemPro (registered trademark)-34 SFM, manufactured by Life Technologies) 1×10 ⁵ ES cells were dispensed into a low-adhesion U-bottom 96-well plate containing a mixture of 0.5 μM Shield1 (manufactured by Clontech) (hereinafter referred to as “oocyte differentiation induction medium”). were transferred and cultured for 5 days to induce differentiation into immature oocytes. FIG. 1 shows the results of observation under a confocal microscope (manufactured by Carl Zeiss, model number: Zeiss LSM 700). In FIG. 1, the term "oocyte-forming genes" refers to eight genes of FIGLA, NOBOX, SOHLH1, LHX8, SUB1, STAT3, TBPL2 and DYNLL1. "Oocyteigenic gene expression OFF" refers to cells before addition of medium containing Shield1, and "Oocyteigenic gene expression ON" refers to cells after addition of medium containing Shield1 and culturing for 5 days.

From FIG. 1, the cells in which the expression of the oocyte formation gene is OFF formed colonies, and almost no fluorescence of ECFP (Stella-ECFP) expressed under the expression control of Stella was observed. In contrast, in cells in which the expression of the oocyte-forming gene is ON, the cells exist separately, and the strong fluorescence of Stella-ECFP was detected, suggesting that they were induced to differentiate into immature oocytes. was done.

(Preparation of mature oocytes)
Then, after culturing for 5 days after transfection, a single colony of ES cells (1×10 ⁵ cells) selected with puromycin was fertilized with 3×10 ⁴ cells after fertilization with the above-mentioned oocyte differentiation medium. Aggregates were prepared by mixing with 5-day-old female mouse-derived ovarian somatic cells and cultured for 2 days. Ovarian somatic cells were previously isolated from the ovaries obtained by removing the ovaries from 12.5-day-old female mice after fertilization using the method described in Non-Patent Document 1 and the like. The aggregates were then transferred onto a Transwell-COL membrane (manufactured by Coaster) and cultured for 28 days in the above oocyte differentiation induction medium. FIG. 2 shows the results of observing the expression of each marker in aggregates on days 2, 6, 8, 10 and 12 from the start of culture under a confocal microscope (manufactured by Carl Zeiss, model number: Zeiss LSM 700). In FIG. 2, in the upper row, oocytes were visualized by fluorescence of the germ cell and oocyte marker Stella-ECFP. At the bottom, oocytes were visualized by the fluorescence of the red fluorescent protein mCherry (Npm2-mCherry) expressed under the control of Nucleoplasmin 2 (Npm2), an activated (mature) oocyte marker.

From FIG. 2, the fluorescence of Stella-ECFP was continuously observed in the cells throughout the culture period. On the other hand, Npm2-mCherry fluorescence was observed on day 8 after the start of culture (see the arrow at the bottom of "Day 8" in FIG. 2), indicating that maturation of the oocytes was progressing.

Individual follicles were also manually isolated using a sharpened tungsten needle on day 28 of culture. The isolated follicles had secondary follicular structures. By culturing the follicles under the culture conditions in the "in vitro growth period" and "in vitro maturation period" described in Non-Patent Document 1, the follicles were matured into second meiotic metaphase eggs via antral follicles.

[Example 2]
Using mouse iPS cells, the induction of differentiation into oocytes was examined in the same manner as ES cells.

(Vector transfection)
Mouse BVSC iPS cells produced by the virus buster established in the paper of Non-Patent Document 1 were used as iPS cells. The PB-CAG-DD vector containing the five genes constructed in Example 1 was transfected into mouse BVSC iPS cells simultaneously with the hyperactive PBase (hypBase) plasmid using Lipofectamine 2000. Single colonies were grown after puromycin selection for 5 days in serum-free medium supplemented with 2i and LIF.

(Differentiation induction into immature oocytes)
The vector-transfected iPS cells were cultured for 5 days in the same manner as in Example 1 to induce differentiation into immature oocytes.

(Preparation of mature oocytes)
Then, after the transfection, cultured for 5 days, puromycin-selected iPS cells (1×10 ⁵ cells) were placed in the above-mentioned oocyte differentiation induction medium, and 3×10 ⁴ cells of 12.5 days old after fertilization. Aggregates were prepared by mixing with female mouse-derived ovary somatic cells and cultured for 2 days. The aggregates were then transferred onto a Transwell-COL membrane (manufactured by Coaster) and cultured for 21 days in the above oocyte differentiation medium. Fig. 3 shows the results of observing the expression of Stella-ECFP in aggregates on day 21 from the start of culture under a confocal microscope (manufactured by Carl Zeiss, model number: Zeiss LSM 700). In FIG. 3, the image on the left is a bright-field image, and the image on the right is a fluorescence image in which oocytes are visualized by the fluorescence of Stella-ECFP, which is a germ cell and oocyte marker.

From FIG. 3, the fluorescence of Stella-ECFP was observed in the cells on the 21st day after the start of culture. From this, it was confirmed that iPS cells could also be induced to differentiate into oocytes in the same manner as ES cells.

[Example 3]
(Identification of key factors in oocyte formation genes)
In order to identify genes that are important factors among oocyte formation genes, a total of 26 types of genes were combined using the same method as in Example 1 so as to obtain the combinations of genes shown in the left diagram of FIG. A vector was constructed. Then, using the same method as in Example 1, mouse ES cells (Blimp1-mVenus: Stella-ECFP: Npm2-mCherry (BVSCNmC)) were transfected with each vector. Using the same method as in Example 1, the transfected ES cells were cultured for 5 days to induce differentiation into immature oocytes. Then, after culturing for 5 days after transfection, single-cell-derived ES cells (1×10 ⁵ cells) selected with puromycin were fertilized with 3×10 ⁴ cells after fertilization. Aggregates were prepared by mixing with ovarian somatic cells derived from 5-day-old female mice and cultured for 2 days. Then, the obtained aggregates were cultured for 21 days using the same method as in Example 1. After culture, the number of oocytes formed from each cell line was counted. Furthermore, the oocyte area was calculated from the fluorescence area of the blue fluorescent protein CFP expressed downstream of the Stella gene. These results are shown in FIG.

From FIG. 4, among the oocyte formation genes, four genes consisting of FIGLA, NOBOX, LHX8 and TBPL2 are important factors for induction of differentiation from pluripotent stem cells to immature oocytes and maturation of oocytes. It became clear that It was also revealed that the efficiency of oocyte formation was further improved by introducing the STAT3 gene in addition to the four types of genes, FIGLA, NOBOX, LHX8 and TBPL2.

According to the method for inducing immature oocytes of the present embodiment, immature oocytes can be easily induced from cells having the ability to differentiate into oocytes such as pluripotent stem cells in a shorter period of culture than conventionally. can do. According to the method for producing mature oocytes of the above aspect, a large amount of mature oocytes are produced from cells having the ability to differentiate into oocytes such as pluripotent stem cells in a shorter period of culture than conventionally. be able to.

Claims (9)

Four types of genes consisting of FIGLA, NOBOX, LHX8 and TBPL2, or their transcripts or expressed proteins are introduced into at least one cell selected from the group consisting of pluripotent stem cells, epiblast-like cells and primordial germ cells. A method of inducing immature oocytes, comprising: 2. The method for inducing immature oocytes according to claim 1, comprising introducing four genes consisting of FIGLA, NOBOX, LHX8 and TBPL2 into said cells. 3. The method of inducing immature oocytes according to claim 1 or 2, further comprising introducing a STAT3 gene, or its transcript or expressed protein into said cells. 4. The method according to any one of claims 1 to 3, further comprising introducing into the cell one or more genes selected from the group consisting of SOHLH1, SUB1 and DYNLL1, or transcripts or expressed proteins thereof. A method for inducing mature oocytes. 5. The method for inducing immature oocytes according to claim 4, further comprising introducing three genes consisting of SOHLH1, SUB1 and DYNLL1 into said cells. The method for inducing immature oocytes according to any one of claims 1 to 5, wherein the cells are pluripotent stem cells. The expression of the gene is regulated so as to be induced by the presence of an expression inducer,
Proliferating said cells after said introduction;
The method for inducing immature oocytes according to any one of claims 1 to 6, further comprising adding the expression-inducing substance to the medium after the proliferation to induce the expression of the gene. Introducing four types of genes consisting of FIGLA, NOBOX, LHX8 and TBPL2, or their transcripts or expressed proteins into at least one cell selected from the group consisting of pluripotent stem cells, epiblast-like cells and primordial germ cells. and,
co-culturing the cells and ovarian somatic cells after introduction;
A method of producing a mature oocyte, comprising: 9. The method of producing mature oocytes according to claim 8, further comprising introducing a STAT3 gene, or its transcript or expressed protein into said cells.

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